ABSTRACT In a wide variety of human diseases, eosinophils and/or mast cells are thought to play an important pathogenetic role. Several drugs are available to treat affected patients, but they are often ineffective. Thus, the need for novel therapeutics to improve outcomes for these disorders is unquestioned. One promising drug target for this purpose is sialic acid immunoglobulin (Ig)-like lectin 8 (Siglec-8; also known as SAF2). Siglec-8 is uniquely expressed on eosinophils, mast cells and (weakly) basophils late during the maturation process and is not displayed on hematopoietic stem cells and other blood and non-blood cells. This very restricted expression pattern ? together with the observation that unconjugated Siglec-8 antibodies (mAbs) cause apoptosis of eosinophils in vitro ? provides the impetus to develop Siglec-8-directed immunotherapy for patient application. While early clinical trials with a humanized Siglec-8 mAb have been initiated, it is unknown how Siglec-8 is best targeted therapeutically. In vitro data showing Siglec-8 mAbs inhibit mast cell function but do not induce mast cell apoptosis suggest that unconjugated mAbs may have insufficient activity in some Siglec-8-positive disorders. This is reminiscent of the experience with Siglec-3 (CD33) ? the currently most widely targeted Siglec family member ? where unconjugated mAbs have been largely ineffective in the clinic. More potent treatment modalities, e.g. CD3-directed bispecific antibodies (BiAbs), may be required for successful Siglec-8 therapy. Besides treatment modality, our data suggest that the efficacy of Siglec-directed immunotherapy is also affected by the location of the domain it targets. Specifically, in our studies with Siglec-3, we found membrane-proximal binding substantially enhances effector functions of CD3-directed BiAbs. As a first step toward our long-term goal of developing and optimizing Siglec-8-directed immunotherapy for eosinophilic and mast cell disorders, we here propose to use humanized (?Trianni?) mice to generate a panel of diverse, fully human Siglec-8 mAbs and then to carefully compare the efficacy of different treatment modalities (mAbs vs. BiAbs) and to determine the role of membrane-proximal targeting as means to enhance the cytotoxicity of Siglec-8-targeted therapeutics. Our studies are expected to provide critical insight into the principles of how Siglec-8 should be targeted for maximal therapeutic benefit while, at the same time, they generate candidate molecules for further clinical development. Our work may lead to a new treatment option for patients with eosinophilic or mast cell disorders for whom outcomes continue to be unsatisfactory.